Weaving a tight social net: Allogrooming in free-ranging female langurs (Presbytis entellus)

We studied grooming among adults of a one-male multifemale troop of free-ranging Hanuman langurs (Presbytis entellus)living near Jodhpur, India, for 9 years. The 11–13 females devoted about 6% of their day to allogrooming. Adult males, whose tenures averaged 2.2 years, were transient figures in the troop's history, as reflected by their rather peripheral role in the grooming network. Females groomed males 4–40 times more frequently (1006 episodes) than vice versa- (176 episodes). Adult females received 97% of all grooming from other adult females (6655 episodes). Although females exhibited an age- inversed dominance hierarchy, they did not compete for grooming access to particular troop mates. Dyads of all possible rank differences occurred as frequently as expected: 51% of grooming was directed up the hierarchy and 49% down it. Young, high- ranking individuals gave and received significantly more grooming than the oldest, low- ranking females did. The pattern seemed to be influenced by kin selection because of the presumably high degree of female relatedness. They invested most in troopmates with the highest reproductive value, i.e., the youngest individuals. This trend was coupled with a preference of closest kin (mothers and daughters). Reciprocity was the outstanding feature since all adult females groomed and were groomed by all others. Such a tight social net might establish the necessary cohesion during frequent territorial disputes with neighboring troops.     

Suppression of growth defects of α-amylase secreting Escherichia coli by signal sequence fusion

Abstract In a previous study, we have described unusual cross-reactions among monoclonal antibodies (Mabs) to bacteria and in particular to the Inaba and Ogawa serotypes of Vibrio cholerae . In this study, the extent to which the binding sites of both antibodies and antigens overlap has been investigated by competitive binding and idiotypic analysis. The competitive binding data indicate that the cross-reactive binding of the Inaba Mabs to the Ogawa vibrios can be abolished by incubation with higher affinity Ogawa Mabs. However, rabbit antiserum raised against the Inaba series does not react with the Ogawa series, indicating that anti-Inaba Mabs do not share idiotypic determinants with anti-Ogawa Mabs. The results therefore suggest that the two sets of antibodies recognise different determinants which are closely related in spatial terms, and which consequently do not permit simultaneous binding of the two types of monoclonal antibody.     

Time- and media-dependent secondary carotenoid accumulation in Haematococcus pluvialis

The green microalgae Haematococcus pluvialis synthesizes secondary carotenoids after exposure to environmental stress, a process that is used for the biotechnological production of astaxanthin (Ax). This study reports, for the first time, the medium-dependent changes in the carotenoid pattern throughout the cultivation process as well as the exact composition of carotenoids and their fatty acid mono- and diesters using LC-MS. Secondary carotenoid formation started immediately upon exposure to nutrient depletion and high light conditions. Ax and its corresponding mono- and diesters were detected simultaneously. After 15 days of cultivation, no significant changes were detected in carotenoid composition; however, the ratio between carotenoid mono- and diesters still varied. Main carotenoids were identified as Ax linolenate and Ax oleate, but also five adonirubin and one lutein monoester were detected. The influence of three different autotroph media was studied on carotenoid content, which reached a maximum 16.1 mg/g dry weight. The results indicate that media composition has an influence on the ratio of Ax mono- to diester but not on the qualitative composition of secondary carotenoids in H. pluvialis. Beside the pathway via echinenone, canthaxanthin and adonirubin the results indicate that Ax biosynthesis takes place via another route: from beta-carotene via beta-cryptoxanthin, zeaxanthin and adonixanthin.     

Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.     

Intimate cell conjugate formation and exchange of membrane lipids precede apoptosis induction in target cells during antibody-dependent, granulocyte-mediated cytotoxicity

Ab-dependent polymorphonuclear granulocyte (PMN)-mediated cytotoxicity may play an important role in the control of malignant diseases. However, little is known as to which particular pathways are used for the killing of malignant cells by PMN. The production of reactive oxygen intermediates (ROI) has been observed to occur during Ab-dependent, cell-mediated cytotoxicity (ADCC). However, PMN from a patient with chronic granulomatous disease demonstrated strong ADCC against malignant lymphoma cells. Furthermore, the inhibition of ROI production in PMN from healthy donors had no significant effect on ADCC. Therefore, ROI production by the NADPH oxidase of PMN does not appear to be mandatory for PMN-mediated ADCC. Recent data suggest a role for perforins in PMN-mediated cytotoxicity. However, in our assays concanamycin A, an inhibitor of perforin-mediated ADCC by mononuclear cells, had no inhibitory effect on PMN-mediated ADCC. Using electron microscopy we observed that PMN and their target cells intimately interact with the formation of interdigitating membrane protrusions. During PMN and target cell contact there was a mutual exchange of fluorescent membrane lipid dyes that was strongly increased in the presence of tumor-targeting Abs. This observation may be closely related to the recently described process of trogocytosis by lymphocytes. The presence of transient PMN-tumor cell aggregates and the accumulation of PMN with tumor cell-derived membrane lipids and vice versa were associated with effective ADCC as measured by chromium-release or apoptosis induction.     

Moraxella catarrhalis is internalized in respiratory epithelial cells by a trigger-like mechanism and initiates a TLR2- and partly NOD1-dependent inflammatory immune response

Moraxella catarrhalis is an important pathogen in patients with chronic obstructive lung disease (COPD). While M. catarrhalis has been categorized as an extracellular bacterium so far, the potential to invade human respiratory epithelium has not yet been explored. Our results obtained by electron and confocal microscopy demonstrated a considerable potential of M. catarrhalis to invade bronchial epithelial (BEAS-2B) cells, type II pneumocytes (A549) and primary small airway epithelial cells (SAEC). Moraxella invasion was dependent on cellular microfilament as well as on bacterial viability, and characterized by macropinocytosis leading to the formation of lamellipodia and engulfment of the invading organism into macropinosomes, thus indicating a trigger-like uptake mechanism. In addition, the cells examined expressed TLR2 as well as NOD1, a recently found cytosolic protein implicated in the intracellular recognition of bacterial cell wall components. Importantly, inhibition of TLR2 or NOD1 expression by RNAi significantly reduced the M. catarrhalis-induced IL-8 secretion. The role of TLR2 and NOD1 was further confirmed by overexpression assays in HEK293 cells. Overall, M. catarrhalis may employ lung epithelial cell invasion to colonize and to infect the respiratory tract, nonetheless, the bacteria are recognized by cell surface TLR2 and the intracellular surveillance molecule NOD1.     

Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants

Plant biotechnology relies on two approaches for delivery and expression of heterologous genes in plants: stable genetic transformation and transient expression using viral vectors. Although much faster, the transient route is limited by low infectivity of viral vectors carrying average-sized or large genes. We have developed constructs for the efficient delivery of RNA viral vectors as DNA precursors and show here that Agrobacterium-mediated delivery of these constructs results in gene amplification in all mature leaves of a plant simultaneously (systemic transfection). This process, called "magnifection", can be performed on a large scale and with different plant species. This technology combines advantages of three biological systems (the transfection efficiency of A. tumefaciens, the high expression yield obtained with viral vectors, and the post-translational capabilities of a plant), does not require genetic modification of plants and is faster than other existing methods.